Vaccine for protection against lawsonia intracellularis, mycoplasma hyopneumoniae and porcine circo virus

ABSTRACT

The present invention pertains to a vaccine comprising in combination non-live antigens of  Lawsonia intracellularis , of  Mycoplasma hyopneumniae  and Porcine circo virus, and a pharmaceutically acceptable carrier. The invention also pertains to a kit comprising a first container having contained therein non-live antigens of  Lawsonia intracellularis , one or more other containers having contained therein  Mycoplasma hyopneumoniae  and Porcine circo virus antigens and instructions for mixing the antigens of  Lawsonia intracellularis, Mycoplasma hyopneumoniae , and Porcine circo virus to formulate one combination vaccine suitable for systemic vaccination.

REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.12/988,088, filed Oct. 15, 2010, which is a national stage entry under35 U.S.C. §371 of PCT/EP2009/054517, filed on Apr. 16, 2009, whichclaims priority to U.S. Provisional Application No. 61/046,188, filed onApr. 18, 2008, and EP Application No. 08154765.5, filed on Apr. 18,2008. The content of which is hereby incorporated by reference in itsentirety.

The present invention pertains to a vaccine for protection againstLawsonia intracellularis, Mycoplasma hyopneumoniae and Porcine circovirus. Protection in this sense means that the vaccine at least providesa decrease in a negative influence caused by Lawsonia intracellularis,Mycoplasma hyopneumoniae and Porcine circo virus, such negativeinfluence being e.g. tissue damage and/or clinical signs such asdecreased weight gain, diarrhea, coughing, sneezing etc. The presentinvention also pertains to a kit comprising a first container havingcontained therein non-live antigens of Lawsonia intracellularis, one ormore other containers having contained therein Mycoplasma hyopneumoniaeand Porcine circo virus antigens and instructions for mixing theantigens of Lawsonia intracellularis, Mycoplasma hyopneumoniae, andPorcine circo virus to formulate one combination vaccine suitable forsystemic vaccination.

Proliferative enteropathy (PE or PPE, also called enteritis or ileitis)in many animals, in particular pigs, presents a clinical sign andpathological syndrome with mucosal hyperplasia of immature cryptepithelial cells, primarily in the terminal ileum. Other sites of theintestines that can be affected include the jejunum, caecum and colon.Weanling and young adult pigs are principally affected with typicalclinical manifestation of rapid weight loss and dehydration. Naturalclinical disease in pigs occurs worldwide. The disease is consistentlyassociated with the presence of intracellular curved bacteria, presentlyknown as Lawsonia intracellularis.

Mycoplasmal pneumonia of swine caused by the bacterial pathogenMycoplasma hyopneumoniae is a widespread chronic respiratory disease inpigs. Especially young piglets are vulnerable to this, non-fatal,disease. The enzootic pneumonia is a chronic disease that results inpoor feed conversion and stunted growth. The disease is highlycontagious and transmission is usually through direct contact withinfected respiratory tract secretions, e.g. in the form of infecteddroplets after coughing/sneezing. The most problematic consequence ofthis disease is that it predisposes for all kinds of secondaryinfections of the respiratory system. It is estimated that e.g. in theUSA, 99% of all pig farms are infected.

Porcine circo virus is thought to be linked to the post-weaningmultisystemic wasting syndrome (PMWS) observed in young pigs. Thisdisease was encountered for the first time in Canada in 1991.Theclinical signs and pathology were published in 1997 and includeprogressive wasting, dyspnea, tachypnea, and occasionally icterus andjaundice. Porcine circo virus is a small (17 nm) icosahedralnon-enveloped virus containing a circular single stranded DNA genome.PDNS (porcine dermatitis and nephropathy syndrome) is another majorproblem for pig farmers which appeared around the same time as PMWS andwhich is also related to Porcine circo virus. Characteristic of PDNS arered/brown circular skin lesions with haemorrhages, usually on the ears,flanks, legs and hams.

With respect to PE, oral vaccination against Lawsonia intracellularishas shown to be an economically efficient measure to control Ileitis andto allow a better exploitation of the genetic growth potential of thepig (Porcine Proliferative Enteropathy Technical manual 3.0, July 2006;available from Boehringer Ingelheim). Furthermore, oral rather thanparenteral vaccination will reduce the transmission of blood-borneinfections such as PRRS via multi-use needles and the reduction ofinjection site reactions and needles retained in carcasses. It willreduce animal and human stresses, time, labour costs and effort comparedto individual vaccination (McOrist: “Ileitis—One Pathogen, SeveralDiseases” at the IPVS Ileitis Symposium in Hamburg, Jun. 28, 2004).

It is generally understood that the advantage of an attenuated livevaccine approach is that the efficacy of immunity is usually relativelygood, as the host's immune system is exposed to all the antigenicproperties of the organism in a more “natural” manner. Specifically forintracellular bacterial agents such as Lawsonia intracellularis, thelive attenuated vaccine approach is believed to offer the best availableprotection for vaccinated animals, due to a full and appropriate T cellbased immune response. This is in contrast with the variable to poorimmunity associated with subunit or killed vaccine types forintracellular bacteria. This is also specifically true for obligateintracellular bacteria such as Lawsonia intracellularis or the Chlamydiasp, which cause pathogenic infections within mucosa. Studies indicatethat whole live attenuated forms of the intracellular bacteria inquestion are best delivered to the target mucosa, that they are requiredas whole live bacterial forms to produce a fully protective immuneresponse in the target mucosa but also that they are immunologicallysuperior compared to use of partial bacterial components.

It has become a general understanding that a vaccine against Lawsoniaintracellularis needs to be administered orally (see La. TechnicalManual 3.0 as referred to hereabove). This is based on the fact that thebasis of the body's resistance to Ileitis is the local immunity in theintestine, which is the product of cell-mediated immunity and localdefense via antibodies, especially IgA. According to current knowledge,serum antibodies (IgG) do not give any protection simply because they donot reach the gut lumen. It has been demonstrated in studies that oralvaccination produces cell-mediated immunity as well as local productionof IgA in the intestine (Murtaugh, in Agrar-und Veterinär-Akademie,Nutztierpraxis Aktuell, Ausgabe 9, Jun. 2004; and Hyland et al. inVeterinary Immunology and Immunopathology 102 (2004) 329-338). Incontrast, intramuscular administration did not lead to protection.Moreover, next to the general understanding that a successful vaccineagainst intracellular bacteria has to induce cell-mediated immunity aswell as the production of local antibodies, the skilled practitionerknows that only a very low percentage of orally ingested antigens areactually absorbed by the enterocytes, and that the incorporation ofLawsonia intracellularis into the cell is an active process initiated bythe bacterium. Accordingly an inactivated vaccine would provide theintestine with insufficient immunogenic antigen (Haesebrouck et al. inVeterinary Microbiology 100 (2004) 255-268). This is why it is believedthat only attenuated live vaccines induce sufficient cell-mediatedprotection in the intestinal cells (see Technical Manual 3.0 as referredto here-above). At present there is only one vaccine on the market toprotect against Lawsonia intracellularis, viz. Enterisol® Ileitismarketed by Boehringer Ingelheim. This vaccine is a live vaccine fororal administration indeed.

Hitherto combination vaccines of Lawsonia intracellularis have beensuggested in the prior art. However, not many of such combinations haveactually been tested for efficacy. The reason for this is that it isgenerally understood that combination of antigens with antigens ofLawsonia intracellularis can only lead to successful protection if theLawsonia antigens are provided as live (attenuated) cells. In thisrespect, we refer to WO 2005/011731, which also suggests all kinds ofcombination vaccines based on Lawsonia intracellularis. However,regarding the description and claim structure the patent application,the assignee (Boehringer Ingelheim) appears to be convinced thatcombination vaccines are only expected to have a reasonable chance ofsuccess when the Lawsonia antigens are present in the form of livecells. The same is true for WO2006/099561, also assigned to BoehringerIngelheim. Indeed, based on the common general knowledge this is anobvious thought. Combination of live antigens however is notstraightforward given the high chance of interference between theantigens and the difficulty of manufacturing such a live combinationvaccine.

It is an object of the present invention to provide a vaccine to combatLawsonia intracellularis, and at the same time combat one or more otherswine pathogens. To this end a vaccine has been devised that comprisesin combination non-live antigens of Lawsonia intracellularis, Mycoplasmahyopneumoniae and Porcine circo virus, and a pharmaceutically acceptablecarrier. Surprisingly, against the persistent general understanding howto combat Lawsonia intracellularis and that a combination vaccine shouldcomprise live Lawsonia intracelluaris antigens, it was found that byusing non-live Lawsonia intracellularis antigens, in combination withantigens from Mycoplasma hyopneumoniae and Porcine circo virus, avaccine can be provided that protects against Lawsonia intracellularis,Mycoplasma hyopneumoniae and Porcine circo virus.

In general, a vaccine can be manufactured by using art-known methodsthat basically comprise admixing the antigens with a carrier. Typicallythe antigen(s) are combined with a medium for carrying the antigens,often simply referred to as a carrier or “pharmaceutically acceptablecarrier”. Such a carrier can be any solvent, dispersion medium, coating,antibacterial and antifungal agent, isotonic and absorption delayingagent, and the like that are physiologically compatible with andacceptable for the target animal, e.g. by being made i.a. sterile. Someexamples of such carrying media are water, saline, phosphate bufferedsaline, bacterium culture fluid, dextrose, glycerol, ethanol and thelike, as well as combinations thereof. They may provide for a liquid,semi-solid and solid dosage form, depending on the intended mode ofadministration. As is commonly known, the presence of a carrying mediumis not essential to the efficacy of a vaccine, but it may significantlysimplify dosage and administration of the antigen. As such, themanufacturing of the vaccine can take place in an industrial environmentbut also, the antigens could be mixed with the other vaccineconstituents in situ (i.e. at a veterinaries', a farm etc.), e.g.(immediately) preceding the actual administration to an animal. In thevaccine, the antigens should be present in an immunologically effectiveamount, i.e. in an amount capable of stimulating the immune system ofthe target animal sufficiently to at least reduce the negative effectsof a post-vaccination challenge with wild-type micro-organisms.Optionally other substances such as adjuvants, stabilisers, viscositymodifiers or other components are added depending on the intended use orrequired properties of the vaccine.

In an embodiment, the vaccine is in a form suitable for systemicadministration. To applicants surprise it has been found that one caninduce a protection against Lawsonia intracellularis that is comparablewith or even improved with respect to the protection provided by usingthe (single) live vaccine ENTERISOL® ILLEITIS (administered according tothe corresponding instructions), when the combination vaccine accordingto the present invention is administered systemically, i.e. in a waythat it reaches the circulatory system of the body (comprising thecardiovascular and lymphatic system), thus affecting the body as a wholerather than a specific locus such as the gastro-intestinal tract.Systemic administration can be performed e.g. by administering theantigens into muscle tissue (intramuscular), into the dermis(intradermal), underneath the skin (subcutaneous), underneath the mucosa(submucosal), in the veins (intravenous) etc. For systemic vaccinationmany forms are suitable, in particular liquid formulations (withdissolved, emulsified or suspended antigens) but also solid formulationssuch as implants or an intermediate form such as a solid carrier for theantigen suspended in a liquid. Systemic vaccination, in particularparenteral vaccination (i.e. not trough the alimentary canal), andsuitable (physical) forms of vaccines for systemic vaccination have beenknown for more than 200 years. An advantage of this embodiment is thatthe same way of administration can be used that is the current standardfor administering Mycoplasma hyopneumoniae or Porcine circo virusantigens, viz. parenteral, in particular via intramuscular orintradermal injection (in the latter case often needle-less).

In an embodiment the non-live Lawsonia intracellularis antigens areobtained from a carbohydrate containing composition, the carbohydratebeing also found in live Lawsonia intracellularis cells in associationwith the outer cell membrane of these cells. Unexpectedly, by using acarbohydrate containing fraction of Lawsonia intracellularis cells (i.e.a composition containing the carbohydrates as present in live Lawsoniaqintracellularis cells) in the combination vaccine, good protectionagainst PE could be provided. It is noted that for formulating thevaccine a carbohydrate containing composition directly obtained fromLawsonia intracellularis cells could be used but also a compositionderived therefrom, such as a dilution or concentrate of the originalcomposition or an extract, one or more purified components etc. It isnoted that subunits of Lawsonia intracellularis cells have been reportedas antigens in a vaccine for protection against this bacterium. However,these are mainly recombinant proteins and hitherto none of them hasproven to be able and provide good protection. It is also noted that acarbohydrate containing composition, wherein the carbohydrate is alsofound in live Lawsonia intracellularis cells in association with theouter cell membrane of these cells, is known from Kroll et al. (Clinicaland Diagnostic Laboratory Immunology, June 2005, 693-699). However, thiscomposition is used for diagnostics. It has not been tested as aprotective antigen for reasons as stated here-above.

In an embodiment, the carbohydrate containing composition is materialresulting from the killing of Lawsonia intracellularis bacteria. It hasbeen found that a very convenient way of providing the carbohydrate foruse according to the present invention is to simply kill Lawsoniaintracellularis cells and use the material resulting from that as asource for the carbohydrate. To extract the carbohydrate from livingcells could in theory also be done (analogous to the creation of livingghost cells by removing the cell wall) but requires more sophisticatedand thus more expensive techniques. The material as a whole could beused, e.g. a suspension of whole cells or a lysate of Lawsoniaintracellularis cells, or one could purify or even isolate thecarbohydrate out of the material. This method can be performed by usingrelatively simple art-known techniques.

In a preferred embodiment the carbohydrate containing compositioncontains whole cells of killed Lawsonia intracellularis bacteria. Thishas proven to be the most convenient way to provide the carbohydrate asan antigen in the vaccine. Besides, the efficacy of the vaccine is evenfurther increased, possibly since this way of offering the antigen tothe immune system of the target animal better mimics the naturalenvironment of the carbohydrate.

In an embodiment the vaccine comprises an oil in water adjuvantcontaining oil droplets of sub-micrometer size. In general, an adjuvantis a non-specific immunostimulating agent. In principal, each substancethat is able to favor or amplify a particular process in the cascade ofimmunological events, ultimately leading to a better immunologicalresponse (i.e. the integrated bodily response to an antigen, inparticular one mediated by lymphocytes and typically involvingrecognition of antigens by specific antibodies or previously sensitizedlymphocytes), can be defined as an adjuvant. It has been shown thatusing an oil in water adjuvant containing oil droplets of sub-micrometersize provides a very good protection against Lawsonia intracellularis.Indeed, the application of oil in water adjuvants as such is common inconnection with non-live antigens. However, it is generally known thatthe best immunostimulating properties are obtained when the oil dropletsare large in diameter. In particular, oil droplets with a diameterbeneath 1 micrometer are in particular used when it is believed thatsafety is an important issue. In that case, one could use small dropletssince these are known to evoke less tissue damage, clinical signs etc.However, in the case of obtaining protection for a gut associateddisorder via systemic vaccination (as is the case in the presentinvention), one would choose large droplets since one would expect thatthe immune response has to be boosted significantly. In contrast, wefound that using small oil droplets in the composition provided verygood results with respect to protection against Lawsoniaintracellularis.

In an even preferred embodiment, the adjuvant comprises droplets ofbiodegradable oil and droplets of mineral oil, the droplets ofbiodegradable oil having an average size that differs from the averagesize of the droplets of mineral oil. It has been shown that the use of amixture of biodegradable oil and mineral oil provides very good resultswith regard to efficacy and safety. In addition to this, stability ofthe composition is very high, which is an important economic advantage.The stability has proven to be very good, in particular when the average(volume weighed) size of either the biodegradable oil droplets or themineral droplets is below 500 nm (preferably around 400 nm).

The present invention also pertains to a kit comprising a firstcontainer having contained therein non-live antigens of Lawsoniaintracellularis, one or more other containers having contained thereinMycoplasma hyopneumoniae and Porcine circo virus antigens andinstructions for mixing the antigens of Lawsonia intracellularis,Mycoplasma hyopneumoniae, and Porcine circo virus to formulate onecombination vaccine suitable for systemic vaccination. In thisembodiment, a separate container for the Lawsonia intracellularisantigens is provided in a kit containing also the other antigens (whichare either combined in one container as known from the prior art or evenpresent in separate containers that form part of the contents of thekit. An advantage of this embodiment is that the Lawsonia antigens canbe prevented from having interactions with the other antigens untilright before administration of the vaccine. Also, since the antigens arein a separate container less production losses will occur. In anembodiment the Mycoplasma hyopneumoniae and Porcine circo virus antigensare contained in one container, formulated in an oil in water adjuvant.In this embodiment, the Lawsonia antigens can be mixed with the otherones just before use.

The invention will be further explained based on the following examples.

Example 1 describes a method to obtain a substantially protein freecarbohydrate containing composition and a vaccine that is made by usingthis composition.

Example 2 describes an experiment wherein a non-live Lawsoniaintracellularis vaccine is compared with the vaccine currently on themarket and an experimental vaccine comprising subunit proteins ofLawsonia intracellularis.

Example 3 describes an experiment wherein two different vaccinescomprising non-live Lawsonia intracellularis antigens are compared withthe live vaccine currently on the market.

EXAMPLE 1

In this example a method is described to obtain a substantially proteinfree carbohydrate composition associated with the outer cell membrane ofLawsonia intracellularis cells and a vaccine that can be made using thiscomposition. In general, a carbohydrate is an organic compound thatcontains carbon, hydrogen, and oxygen, usually in the ratio 1:2:1.Examples of carbohydrates are sugars (saccharides), starches,celluloses, and gums. Usually they serve as a major energy source in thediet of animals. Lawsonia intracellularis is a gram negative bacterium,which thus contains an outer membrane that is not constructed solely ofphospholipid and protein, but also contains carbohydrates, in particularpolysaccharide (usually polysaccharides such as lipopolysaccharide,lipo-oligosaccharide, or even non-lipo polysaccharides).

Carbohydrate Fraction for Vaccine Preparation

Twenty milliliters of buffered water (0.04 M PBS, phosphate bufferedsaline) containing Lawsonia intracellularis cells at a concentration of3.7E8 (=3.7×10⁸) cells/ml was taken. The cells were lysed by keepingthem at 100° C. for 10 minutes. Proteinase K (10 mg/ml) in 0.04 M PBSwas added to a final concentration of 1.7 mg/ml. This mixture wasincubated at 60° C. for 60 minutes in order to degrade all proteins andkeep the carbohydrates intact. Subsequently, the mixture was incubatedat 100° C. for 10 minutes to inactivate the Proteinase K. The resultingmaterial, which is a carbohydrate containing composition, in particularcontaining the carbohydrates as present in live Lawsonia intracellularisbacteria in association with their outer cell membrane (see paragraphbelow), was stored at 2-8° C. until further use. The composition wasformulated in DILUVAC FORTE adjuvant which also serves as a carrier forthe antigens. This adjuvant (see also EP 0 382 271) comprises 7.5 weightpercent vitamine E acetate droplets with an average volume weighted sizeof approximately 400 nm, suspended in water and stabilized with 0.5weight percent of TWEEN 80 (polyoxyethylene sorbitan mono-oleate). Eachmilliliter vaccine contained material that had been extracted from 1.2E8Lawsonia intracellularis cells.

Immune Precipitation of Lawsonia Carbohydrate Antigens

Two batches of monoclonal antibodies (MoAb's) raised against whole cellLawsonia intracellularis were precipitated with saturated Na₂SO₄ at roomtemperature according to standard procedures. The precipitate waspelleted by centrifugation (10.000 g for 10 minutes). The pellet waswashed with 20% Na₂SO₄ and resuspended in 0.04 M PBS. TYLOSYL activatedDYNAL beads (DynaBeads, DK) were pre washed with 0.1 M NaPO₄ (pH 7.4),according the manual of the manufacturer. Of each batch of MoAb's 140 μgwas taken and added to 2E8 pre washed beads and incubated overnight at37° C. The beads were pelleted by centrifugation and non-bound MoAb'swere removed by aspiration of the supernatant. Spectrophotometricalmeasurements showed that between 20 and 35% of the added MoAb's hadbound to the beads. Two batches of 1 ml Lawsonia intracellularis cells(3.7E8/ml) in 0.04 M PBS were sonicated for 1 minute. The resulting celllysates were added to the TYLOSYL activated beads—monoclonal complexesand incubated overnight at 4° C. The TYLOSYL activated beads—monoclonalcomplexes were washed three times with 0.1 M NaPO₄ (pH 7.4). The boundcompounds were eluted by washing the beads in 0.5 ml 8M urea in 0.04 MPBS (E1); 0.5 ml 10 mM Glycine pH 2.5 (E2); and 0.5 ml 50 mM HCl (E3),in a sequential manner. After elution E2 and E3 were neutralized witheither 100 μl and 200 μl 1 M Tris/HCl (pH8.0).

Samples were taken from each step and loaded onto SDS-PAGE gels. Gelswere stained using Commassie Brilliant Blue (CBB) and Silver staining orblotted. The blots were developed using the same MoAb's as mentionedhere-above. Inspection of the gels and blots showed that the MoAb'srecognized bands with an apparent molecular weight of 21 and 24 kDa thatwere not seen on the CBB gels but were visible on de Silver stainedgels. Also, it was established that the fraction of the cells that boundto the MoAb's was Proteinase K resistant. Thus, based on these resultsit can be concluded that this fraction contains carbohydrates (namely:all protein is lysed, and sonified DNA fractions will not show as aclear band in a Silver stain), and that the carbohydrates are inassociation with (i.e. forming part of or being bound to) the outer cellmembrane of Lawsonia intracellularis (namely: the MoAb's raised againstthis fraction also recognized whole Lawsonia intracellularis cells).Given the fact that Lawsonia intracellularis is a gram-negativebacterium, the carbohydrate composition is believed to comprisepolysaccharide(s).

EXAMPLE 2

This experiment was conducted to test a convenient way to formulate thecarbohydrate antigen in a vaccine, viz. via a killed whole cell (alsoknown as bacterin). As controls the commercially available vaccineENTERISOL® ILEITIS and an experimental subunit vaccine comprisingprotein subunits were used. Next to this unvaccinated animals were usedas a control.

Experimental Design of Example 2

An inactivated whole cell vaccine was made as follows. Live Lawsoniaintracellularis cells derived from the intestines of pigs with PPE weregathered. The cells were inactivated with 0.01% BPL(beta-propiolactone). The resulting material, which inherently is anon-live carbohydrate containing composition in the sense of the presentinvention (in particular since it contains the carbohydrates as presentin live Lawsonia intracellularis bacteria in association with theirouter cell membrane), was formulated in Diluvac forte adjuvant (seeExample 1) at a concentration of approximately 2.8×10⁸ cells per mlvaccine.

The subunit vaccine contained recombinant P1/2 and P4 as known from EP1219711 (the 19/21 and 37 kDa proteins respectively), and therecombinant proteins expressed by genes 5074, 4320 and 5464 as describedin WO2005/070958. The proteins were formulated in Diluvac forteadjuvant. The vaccine contained approximately 50 μgrams of each proteinsper milliliter.

Forty 6-week-old SPF pigs were used. The pigs were allotted to 4 groupsof ten pigs each. Group 1 was vaccinated once orally (at T=0) with 2 mllive “ENTERISOL® ILEITIS” (Boehringer Ingelheim) according to theinstructions of the manufacturer. Group 2 and 3 were vaccinated twiceintramuscularly (at T=0 and T=4w) with 2 ml of the inactivated Lawsoniawhole cell vaccine and the recombinant subunit combination vaccine asdescribed here-above, respectively. Group 4 was left as unvaccinatedcontrol. At T=6w all pigs were challenged orally with homogenized mucosainfected with Lawsonia intracellularis. Subsequently all pigs were dailyobserved for clinical signs of Porcine Proliferative Enteropathy (PPE).At regular times before and after challenge serum blood (for serology)and faeces (for PCR) were sampled from the pigs. At T=9w all pigs wereeuthanized and necropsied. Histological samples of the ileum were takenand examined microscopically.

The challenge inoculum was prepared from infected mucosa: 500 grams ofinfected mucosa (scraped from infected intestines) were thawed and mixedwith 500 ml physiological salt solution. This mixture was homogenized inan omnimixer for one minute at full speed on ice. All pigs werechallenged orally with 20 ml challenge inoculum at T=6w.

At T=0, 4, 6, 7, 8 and 9w a faeces sample (gram quantities) and a serumblood sample of each pig was taken and stored frozen until testing. Thefaeces samples were tested in a quantitative PCR (Q-PCR) test andexpressed as the logarithm of the amount found in picograms (pg). Serumsamples were tested in the commonly applied IFT test (immuno fluorescentantibody test to detect antibodies against whole Lawsoniaintracellularis cells in the serum). For histological scoring a relevantsample of the ileum was taken, fixed in 4% buffered formalin, routinelyembedded and cut into slides. These slides were stained withHematoxylin-Eosin (HE stain) and with an immunohistochemical stain usinganti-Lawsonia intracellularis monoclonal antibodies (IHC stain). Theslides were examined microscopically. The histology scores are asfollows:

HE Stain:

no abnormalities detected score = 0 doubtful lesion score = ½ mildlesions score = 1 moderate lesions score = 2 severe lesions score = 3

IHC Stain:

no L. intracelluaris bacteria evident score = 0 doubtful presence ofbacteria score = ½ presence of single/small numbers of bacteria in theslide score = 1 presence of moderate numbers of bacteria in the slidescore = 2 presence of large numbers of bacteria in the slide score = 3

All data were recorded for each pig individually. The score per groupwas calculated as the mean of the positive animals for the differentparameters after challenge. The non-parametric Mann-Whitney U test wereused to evaluate the statistical significance (tested two-sided andlevel of significance set at 0.05).

Results of Example 2 Serology

Before first vaccination all pigs were seronegative when tested for IFTantibody titres. After vaccination with the whole cell bacterin (group2) pigs developed high IFT antibody titres whereas the controls and thepigs vaccinated with the subunit vaccine remained negative untilchallenge (Table 1). Two of the ENTERISOL vaccinated pigs (group 1)developed moderate IFT titres whereas all other pigs in this groupremained seronegative. After challenge all pigs developed high IFTantibody titers. Mean results are depicted in table 1 (with the useddilution, 1.0 was the detection level on the lower side).

TABLE 1 Mean IFT antibody titres (2log) of pig serum after vaccinationand challenge Group T = 0 weeks T = 4 weeks T = 6 weeks T = 9 weeks 1<1.0 1.1 1.7 >11.4 2 <1.0 3.7 >11.8 >12.0 3 <1.0 <1.0 <1.0 >11.6 4 <1.0<1.0 <1.0 >12.0

Real-Time PCR on Faeces Samples

Before challenge all faeces samples were negative. After challengepositive reactions were found in all groups. Group 1 (p=0.02), group 2(p=0.01) and group 3 (p=0.03) had a significantly lower shedding levelcompared to the control. A post-challenge overview is given in table 2.

TABLE 2 Mean results of PCR on faeces samples (log pg) after vaccinationand challenge T = 6 T = 7 T = 8 T = 9 Total Group weeks weeks weeksweeks post-challenge 1 0 1.3 3.6 1.8 6.3 2 0 0.8 2.8 1.9 5.5 3 0 0.5 3.82.0 5.9 4 0 0.8 4.9 4.9 10.0

Histology Scores

Group 2 had the lowest histology HE score (p=0.05), IHC score (p=0.08)and total histology score (p=0.08). The other groups had higher scoresand were not significantly different from the control group. See table3.

TABLE 3 Mean histology score for the ileum. Group HE score IHC scoreTotal score 1 1.8 1.5 3.3 2 1.3 1.5 2.7 3 1.8 1.6 3.4 4 2.4 2.3 4.7Conclusions with Regard to Example 2

From the results it can be concluded that the non-live whole cellLawsonia intracellularis vaccine which inherently contains thecarbohydrate as found also in association with the outer membrane oflive Lawsonia intracellularis cells, induced at least partialprotection. All parameters studied and histology scores weresignificantly or nearly significantly better compared to the controls.

EXAMPLE 3

This experiment was conducted to test a vaccine comprising asubstantially protein free carbohydrate containing composition asantigen. A second vaccine to be tested contained in addition to killedwhole cells of Lawsonia intracellularis, antigens of Mycoplasmahyopneumoniae and Porcine circo virus (the “combi” vaccine). As acontrol the commercially available ENTERISOL® ILEITIS vaccine was used.Next to this, unvaccinated animals were used as a second control.

Experimental Design of Example 3

The vaccine based on a substantially protein free carbohydratecontaining composition was obtained as described under Example 1.

The experimental combi vaccine contained inactivated Lawsoniaintracellularis whole cell antigen (see Example 2 for the used method ofproviding the inactivated bacteria) at a level of 1.7×10⁸ cells/ml. Nextto this it contained inactivated PCV-2 antigen (20 μgrams of the ORF 2encoded protein of PCV 2 per ml; the protein being expressed in a baculovirus expression system as commonly known in the art, e.g. as describedin WO 2007/028823) and inactivated Mycoplasma hyopneumoniae antigen (thesame antigen in the same dose as is known from the commerciallyavailable vaccine PORCILIS MHYO®, obtainable from Intervet, Boxmeer, TheNetherlands). The antigens were formulated in a twin emulsion adjuvant“X”. This adjuvant is a mixture of 5 volume parts of adjuvant “A” and 1volume part of adjuvant “B”. Adjuvant “A” consists of mineral oildroplets with an approximate average (volume weighed) size around 1 μm,stabilised with TWEEN 80 in water. Adjuvant “A” comprises 25 weight % ofthe mineral oil and 1 weight % of the Tween. Rest is water. Adjuvant “B”consists of droplets of biodegradable vitame E acetate with anapproximate average (volume weighed) size of 400 nm, stabilised alsowith TWEEN 80. The adjuvant “B” comprises 15 weight % of vitamine Eacetate and 6 weight % of TWEEN 80, rest is water.

Sixty-four 3-day-old SPF piglets were used. The pigs were allotted tofour groups of 14 piglets and one group of 8 piglets (Group 4). Group 1was vaccinated intramuscularly at 3 days of age with 2 ml of the combivaccine, followed by a second vaccination at 25 days of age. Group 2 wasvaccinated intramuscularly once with 2 ml combi vaccine at 25 days ofage. Group 3 was vaccinated orally with 2 ml ENTERISOL®ILEITIS(Boehringer Ingelheim) at 25 days of age according to prescriptions.Group 4 was vaccinated intramusculary at 3 and 25 days of age with 2 mlof the non-protein carbohydrate vaccine. Group 5 was left unvaccinatedas a challenge control group. At 46 days of age all pigs were challengedorally with homogenized infected mucosa. Subsequently all pigs weredaily observed for clinical signs of Porcine Proliferative Enteropathy(PPE). At regular times before and after challenge serum blood andfaeces samples were taken from the pigs for serology and PCRrespectively. At 68 days of age all pigs were euthanized and post-mortemexamined. The ileum was examined histologically.

The other issues in the experimental design were the same as describedin Example 2, unless indicated otherwise.

Results of Example 3 Serology 1—Lawsonia

Before first vaccination all pigs were seronegative for IFT antibodytitres. After vaccination with the combi vaccine (groups 1 and 2) andthe non-protein carbohydrate vaccine (group 4), many pigs developed IFTantibody titres whereas the controls and the pigs vaccinated withENTERISOL remained seronegative until challenge. After challenge allpigs (except two in the ENTERISOL group) developed IFT antibody titres.For an overview of the mean values obtained, see table 4 (due to thehigher dilution when compared to example 2, the detection level was4.0).

TABLE 4 Mean IFT Lawsonia antibody titres (2log) of pig serum aftervaccination and challenge Group T = 3 days T = 25 days T = 46 days T =67 days 1 <4.0 <4.0 7.9 10.3 2 <4.0 <4.0 4.8 9.8 3 <4.0 <4.0 <4.0 8.5 4<4.0 <4.0 6.9 10.6 5 <4.0 <4.0 <4.0 9.02—Mycoplasma hyopneumoniae

With respect to Mhyo, at the start of the experiment as well as day ofbooster (25-day-old) all pigs were seronegative for Mhyo. After boostervaccination group 1 developed high Mhyo antibody titres, at the samelevel of those obtained with the commercially available prime-boostvaccine PORCILIS MHYO®. The results are given in Table 5 below.Apparently, under these circumstances (given intramuscularly) only whena booster vaccination is given the antibody titres at 46 days are abovedetection level (6.0 for the method used). It is known however that asingle shot vaccination with Mhyo antigens may provide sufficientprotection, in particular when administered intradermally (see e.g. WO2007/103042).

TABLE 5 Mean IFT Mhyo antibody titres (2log) of pig serum aftervaccination Group T = 3 days T = 25 days T = 46 days 1 Below detectionBelow detection 8.3 level level 2 Below detection Below detection Belowdetection level level level 5 Below detection Below detection Belowdetection level level level

3—Porcine Circo Virus

With respect to PCV, at 3-day-old the piglets had high maternallyderived PCV antibody titres. At day of booster (25-day-old) thevaccinates (group 1) had a similar titre compared to group 2 and thecontrol group. The PCV titre at 25-day-old was slightly lower comparedto the titre at 3-day-old. After the vaccination at 25-day-old thetitres of group 1 (2 vaccinations: at day 3 and 25) and group 2 (onevaccination at day 25) remained at a high level whereas control pigletsshowed a normal decrease in maternally derived antibodies. The PCVtitres obtained are comparable to the titres obtainable with a singlevaccine containing the same antigen (e.g. Intervet's Circumvent PCV,which vaccine provides very good protection against PCV). For anoverview of the mean values, see the table given below.

TABLE 6 Mean IFT PCV antibody titres (2log) of pig serum aftervaccination Group T = 3 days T = 25 days T = 46 days 1 11.5 9.6 10.1 212.1 9.5 10.8 5 10.9 9.1 7.0

Real-Time PCR on Faeces Samples

Three weeks after challenge, pigs of group 1, 2 and 4 had less Lawsonia(DNA) in their feces compared to groups 3 and 5. Only the differencesbetween group 1 and 3 (ENTERISOL) and group 4 and 3 were statisticallysignificant (p<0.05, Mann-Whitney U test). For the mean results, seetable 7.

TABLE 7 Mean results of PCR on faeces samples (log pg) after vaccinationand challenge Group Mean value 1 1.0 2 1.2 3 2.0 4 0.6 5 1.8

Histological Scores

Histology scores of group 1 and 4 were significantly lower compared tothose of groups 3 and 5 (p<0.05, two-sided Mann-Whitney U test (seetable 8). The number of pigs with confirmed PPE were 2/13 in group 1,6/12 in group 2, 12/14 in group 3, 2/7 in group 4 and 12/14 in thecontrol group 5. Groups 1 and 4 had a significantly lower incidence ofPPE compared to groups 3 and 5 (p<0.05, two-sided Fischers' exact test).

TABLE 8 Mean histology score for the ileum. Group HE Score IHC ScoreTotal Score 1 0.4 0.6 1.0 2 0.7 0.7 1.4 3 1.6 1.4 3.0 4 0.4 0.4 0.8 51.9 1.5 3.4

Conclusion of Example 3

From the results it can be concluded that the carbohydrate outer cellmembrane antigen offers a relatively good protection against ileitis. Itis also found that the whole cell Lawsonia bacterin is a good means ofoffering the carbohydrate antigen in a vaccine to combat ileitis.Moreover, given the fact that the combination vaccine provided titresfor

Mhyo and PCV antibodies to a level comparable with the levels obtainablewith available single vaccines that are adequate to combat thesemicro-organisms, it has been demonstrated that a combination vaccinecomprising non-live Lawsonia intracellularis antigens in combinationwith Mhyo and PCV antigens is suitable to combat Lawsoniaintracellularis as well as Mycoplasma hyopneumoniae and Porcine circovirus.

1-9. (canceled)
 10. A method for protecting a swine against virulentchallenge by Lawsonia intracellularis, Mycoplasma hyopneumoniae andPorcine Circo Virus infection comprising systemically administering tothe swine a non-live combination vaccine comprising an immunogenicallyeffective amount of non-live antigens of Lawsonia intracellularis,Mycoplasma hyopneumonia and Porcine Circo Virus, and a carrier, whereinthe non-live Lawsonia intracellularis antigens comprise a carbohydratefound in live Lawsonia intracellularis cells in association with theouter cell membrane of those cells.
 11. The method according to claim10, wherein the Lawsonia intracellularis antigens comprise materialresulting from the killing of Lawsonia intracellularis bacteria.
 12. Themethod according to claim 11, wherein the Lawsonia intracellularisantigens comprise killed whole cells of Lawsonia intracellularisbacteria.
 13. The Method according to claim 10, wherein the vaccinecomprises an oil in water adjuvant containing oil droplets ofsub-micrometer size.
 14. The method according to claim 13, wherein theadjuvant comprises droplets of biodegradable oil and droplets of mineraloil, the droplets of biodegradable oil having an average size thatdiffers from the average size of the droplets of mineral oil.